In recent years, the contamination of the human blood supply with a variety of life-threatening and debilitating viruses, such as retroviruses, including HIV (AIDS) has generated a need for a rapid, inexpensive method to purify DNA from human tissues so as to detect viral DNA in human blood. Tests to detect viruses in blood traditionally detect antibodies specific for such viruses. If the nucleic acid sequence of a pathogenic virus is known, the presence of that virus in cells and tissues can be detected in DNA purified from the biological sample. However, it is easier to rely on serological tests of the virus' presence than a test for detecting the viral-specific DNA. Serological tests detect a subject's antibody production against the viral agent. Tests for detecting the presence of antibodies are more efficient and easier to perform than tests for detecting the presence of viral DNA in tissues. Purification of cellular DNA to detect the presence of viral-specific DNA incorporated into genomic DNA requires hours of complicated cell/tissue lysate preparation and treatment.
These tests, however, fail to detect the presence of the virus early in the infection, before an individual mounts a detectable antibody response to the viral agent. Tests to directly detect viruses are also available but they are not practical, not easy to perform, and they are expensive. They include virus culture. Virus culture is not practical for mass screening of blood because up to a month is required to complete the culture. Alternatively, upon purification of DNA from tissues and cells, conventional nucleic acid hybridization techniques for the detection of viral nucleic acid (Ausubel, F. M., et al. eds., Current Protocols in Molecular Biology, John Wiley & Sons (1987)) could be carried out. Although the sensitivity of such hybridization techniques may be enhanced by amplifying the amount of viral DNA with the polymerase chain reaction (PCR), (Mullis, K. et al. Methods in Enzymology, 155:335 (1987)) which amplifies DNA in a sequence-specific manner, nonetheless, the DNA, if it is desired to use a large starting amount, must be first purified substantially free of proteins and RNA. Current methods for purifying DNA are too costly for use in mass screening. For mass screening purposes which involve testing of substantially large amounts of purified DNA for the presence of viral specific DNA sequences, a less involved method for sample preparation would be desirable. Clearly, a method for detecting the presence of viral DNA in the absence of detectable antibody is important to ensure that a blood supply is free from virus contamination.
Purifying DNA from tissue or cell samples is complicated, time consuming, and requires chemicals and equipment that are hazardous and/or expensive. Most current methods for DNA preparation use traditional organic solvent extractions and/or absorption columns. In general, optimal recovery of DNA from biological samples is achieved by a phenol extraction followed by ethanol precipitation. This requires training and technical skills so that DNA is obtained substantially free of proteins and RNA.
Clinically useful applications of DNA purification from human tissues, for example, involve the detection of disease-causing, viral-specific genomes incorporated into human chromosomes, such as human immunodeficiency virus (HIV). Another useful application is the detection of disease causing genes, such as cystic fibrosis, sickle cell anemia, and Duchenne muscular dystrophy.
Kits are now available which allow DNA isolation from tissues without the use of phenol/chloroform extraction. For example, A.S.A.P..TM. Genomic DNA Isolation Kit, Boehringer Mannheim Biochemicals, Indianapolis, Ind.; and The Extractor.TM., Molecular Biosystems, Inc., San Diego, Calif. These kits employ an ion exchange column to retain DNA on the basis of DNA's electrical charge. A disadvantage of these kits is that the entire procedure from cell lysis to elution of purified DNA requires two to four hours for most samples.
Known procedures for DNA purification from whole blood require cumbersome and time consuming steps for cell lysate preparation. The multitude of steps creates a greater potential for specimen confusion and cross contamination. For example, to obtain purified cellular DNA, including viral-specific DNA, from a sample of whole blood, the red blood cells must first be separated from the nucleated white blood cells which contain the DNA. A typical method for preparing specimens of whole blood to purify DNA involves first purifying the mononuclear cells by banding in a density gradient such as ficoll hypaque (Pharmacia, Inc.) washing, then lysing the cells. The cell purification step is necessary because hemoglobin is reported to interfere with the PCR amplification. The isolated mononuclear cells are washed twice with phosphate buffered saline (PBS), then resuspended in 1 ml of PBS. A smear of the cell suspension is made and stained with Wright stain. The proportion of mononuclear cells is consistently found to be greater than 95%. A white cell count is then determined in a Coulter counter on an aliquot of each cell suspension. The cells are then pelleted and lysed by a quick lysis method to give a minimum cell concentration of 3.times.10.sup.6 cells per ml. Proteinase K is added to a final concentration of 120 micrograms/ml and the lysates are incubated at 60.degree. C. for 1 hour. The proteinase K is then inactivated by a 10-minute incubation at 95.degree. C.
Mass screening of the human blood supply would require a mass scale-up of a traditional DNA purification method to detect viral-specific DNA. The cost would be very high for scaling up these methods to purify DNA obtained from either large numbers of samples or large sample volumes collected from a large portion of the population. Accordingly, it is desirable to have a method for rapidly, simply, and inexpensively obtaining purified DNA from small or large volumes or numbers of samples of donated human blood or other tissues.
Furthermore, it would be beneficial if such a simple procedure suitable for rapidly purifying DNA yielded the DNA substantially free of contaminants that can interfere with hybridization techniques or the polymerase chain reaction. Such contaminants include RNA, heparin, detergents, and large amounts of some proteins, like hemoglobin. It would be further desirable for this simple method to yield large amounts of DNA that can be examined for the presence of a single copy of a targeted sequence using the PCR.
Therefore, there is a need for a convenient and reliable technique for purifying large amounts of DNA from biological tissue or cell samples that requires less time than current techniques and does not require organic, hazardous, or expensive reagents. A technique is also needed which can be inexpensively and easily scaled up or down, does not require prior separation of cells from the sample, such as red blood cells, and that yields purified DNA that is substantially free of RNA, proteins, and other contaminants interfering with detection of specific DNA sequences by hybridization and amplification techniques, including polymerase chain reaction techniques.